The long term objective of this research application is to examine the epigenetic mechanisms that participate in human cancer, and to determine how distinct genetic mutations cooperate and/or influence epigenetic control. Cytosine methylation of CpG dinucleotides in gene regulatory regions is an important epigenetic mechanism involved in gene silencing, whereas, mutations in critical transcription factors, such as p53, is an example of a genetic mechanism involved in silencing target genes. We provide evidence that aberrant cytosine methylation and p53 mutation can cooperate to silence genes with tumor suppressor function. We hypothesize pharmacologic strategies that target epigenetic and genetic lesions can synergize to transcriptionally reprogram cells to a near normal state that will result in efficient tumor cell kill or reversal of malignant properties. Three Specific Aims are proposed to address the hypothesis and investigate in greater detail the cooperation between genetic and epigenetic mechanisms in the control of gene expression. Studies will focus on human breast cancer;however, the general rules identified will likely extend to other human cancers. 1) Determine if p53 plays a role in the maintenance of the unmethylated state of target gene promoters. 2) Define further the mechanism by which reversal of genetic and epigenetic lesions synergize to reactivate silenced genes. 3) Identify new genes that are controlled by 5-methylcytosine in breast cancer.